Original Article
Biomol Ther 25(2), 165-170 (2017)
Chemical Constituents Identified from Fruit Body of Cordyceps bassiana and Their Anti-Inflammatory Activity Wonse Suh1,†, Gyeongsug Nam2,†, Woo Seok Yang2,†, Gi-Ho Sung3, Sang Hee Shim4,* and Jae Youl Cho2,* School of Biotechnology, Yeungnam University, Gyeongsan 38541, Department of Genetic Engineering, Sungkyunkwan University, Suwon 16419, 3 Institute for Bio-Medical Convergence, International St. Mary’s Hospital and College of Medicine, Catholic Kwandong University, Incheon 22711, 4 College of Pharmacy, Duksung Women’s University, Seoul 01369, Republic of Korea 1 2
Abstract Cordyceps bassiana is one of Cordyceps species with anti-oxidative, anti-cancer, anti-inflammatory, anti-diabetic, anti-obesity, anti-angiogenic, and anti-nociceptive activities. This mushroom has recently demonstrated to have an ability to reduce 2,4-dinitrofluorobenzene-induced atopic dermatitis symptoms in NC/Nga mice. In this study, we further examined phytochemical properties of this mushroom by column chromatography and HPLC analysis. By chromatographic separation and spectroscopic analysis, 8 compounds, such as 1,9-dimethylguanine (1), adenosine (2), uridine (3), nicotinamide (4), 3-methyluracil (5), 1,7-dimethylxanthine (6), nudifloric acid (7), and mannitol (8) were identified from 6 different fractions and 4 more subfractions. Through evaluation of their anti-inflammatory activities using reporter gene assay and mRNA analysis, compound 1 was found to block luciferase activity induced by NF-kB and AP-1, suppress the mRNA levels of cyclooxygenase (COX)-2 and tumor necrosis factor (TNF)-a. Therefore, our data strongly suggests that compound 1 acts as one of major principles in Cordyceps bassiana with anti-inflammatory and anti-atopic dermatitis activities. Key Words: Cordyceps bassiana, Phytochemical study, 1,9-dimethylguanine, NF-kB, AP-1, Anti-inflammatory activity
INTRODUCTION
C. bassiana was described in China on a carpenterworm larva and connected to unequivocally B. bassiana (Rehner et al., 2011). More recently, C. bassiana was found in Korea and the evidence of the teleomorph-anamorph connection in the Cordyceps specimen was provided, in addition to in vitro production of C. bassiana (Hyun et al., 2013). By systematic analysis of the pharmacological activities of the Cordyceps species to date, anti-oxidative, anti-cancer, anti-inflammatory, anti-diabetic, anti-obesity, anti-angiogenic, and anti-nociceptive activities have been demonstrated (Ng and Wang, 2005; Zhou et al., 2009). Recently, extracts of the artificially cultivated C. bassiana have been reported to inhibit the production of nitric oxide, interleukin (IL)-12, and interferon (IFN)-γ in lipopolysaccharide (LPS)-stimulated macrophages, splenic lymphocytes (Byeon et al., 2011a, 2011b), as well as ameliorating atopic dermatitis symptoms in 2,4-dinitrofluorobenzene-treated NC/Nga mice (Wu et al., 2011). In spite
Cordyceps species are insect-born mushrooms that have been known to possess a variety of biological material. As such, the Cordyceps species has been used for a long time in Chinese and Korean traditional folk medicine as a tonic for longevity, endurance and vitality, and it has been traditionally used as a therapeutic remedy for various diseases, including eczema, skin diseases, chronic bronchitis, asthma, and tuberculosis (Ng and Wang, 2005; Zhou et al., 2009). Cordyceps bassiana is known to be a teleomorph (sexually reproducing form) of Beauveria bassiana. B. bassiana is a ubiquitous fungal pathogen of insect and fungal biocontrol agents widely used against insects (Rehner et al., 2011) and widely used as a medicine for the treatment of infantile convulsions, epilepsy, stroke, sore throat, and many sorts of wounds before being used for insect biocontrol (Madsen et al., 2007). In contrast,
Received Mar 21, 2016 Revised May 5, 2016 Accepted Jun 20, 2016 Published Online Aug 19, 2016
Open Access https://doi.org/10.4062/biomolther.2016.063 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
*Corresponding Authors
E-mail: [email protected] (Shim SH), [email protected] (Cho JY) Tel: +82-2-901-8774 (Shim SH), +82-31-290-7868 (Cho JY) Fax: +82-2-901-8386 (Shim SH), +82-31-290-7870 (Cho JY) † The first three authors contributed equally to this work.
www.biomolther.org
Copyright © 2017 The Korean Society of Applied Pharmacology
165
Biomol Ther 25(2), 165-170 (2017)
A
of its valuable pharmacological activities, no reports on the chemical constituents of C. bassiana have been published to date. Simultaneously, we are trying to develop an anti-atopic dermatitis remedy with this species, and have also performed research to dissect the chemical constituents of C. bassiana in order to identify the active components. Herein, isolation, structure determination, and anti-inflammatory activities of the 8 major compounds from a water extract of C. bassiana fruit body displaying anti-atopic dermatitis activity are described.
MATERIALS AND METHODS Materials
Artificially cultivated Cordyceps bassiana (C. bassiana) (Fig. 1A) were purchased from Mush-Tech (Gangwon, Korea), identified by Prof. Jae Mo Sung (Kangwon National University, Chuncheon, Korea). A voucher specimen (No. 1001) was deposited in the herbarium of Daewoong Pharmaceutical Co. Ltd. (Yongin, Korea). Lipopolysaccharide (LPS, E. coli 0111:B4) and (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bay 11-7082 (BAY), SB203580 (SB), and U0126 (U0) were obtained from Calbiochem (La Jolla, CA, USA). Luciferase constructs containing binding promoters for NF-kB and AP-1 were gifts from Prof. Chung Hae Young (Pusan National University, Pusan, Korea) and Man Hee Rhee (Kyungpook National University, Daegu, Korea). Foetal bovine serum and RPMI 1640 were obtained from GIBCO BRL (Grand Island, NY, USA). RAW264.7 and HEK293 cells were purchased from ATCC (Rockville, MD, USA). All other chemicals were of Sigma grade. A powdered extract of C. bassiana was provided by Daewoong Pharmaceutical Co. Ltd (Yongin, Korea).
Artificially cultivated fruit body of C. bassiana
B Luciferase activity (fold increase)
50
30 20 10
**
**
*
**
0 Nor
0 50 100 NF- B-Luc
Nor
0 50 100 AP-1-Luc
Fig. 1. A photo of an artificially cultivated fruit body of Cordyceps bassiana and the inhibitory activity of its water extract on AP-1 and NF-kB-mediated luciferase. (A) A photo of an artificially cultivated fruit body of Cordyceps bassiana. (B) HEK293 cells co-transfected with plasmid constructs NF-kB-Luc or AP-1-Luc (each 1 mg/ml) and b-gal (as a transfection control) were treated with the water extract of Cordyceps bassiana in the presence or absence of PMA (100 nM). Luciferase activity was measured using a luminometer. *p
Biomol Ther 25(2), 165-170 (2017)
Chemical Constituents Identified from Fruit Body of Cordyceps bassiana and Their Anti-Inflammatory Activity Wonse Suh1,†, Gyeongsug Nam2,†, Woo Seok Yang2,†, Gi-Ho Sung3, Sang Hee Shim4,* and Jae Youl Cho2,* School of Biotechnology, Yeungnam University, Gyeongsan 38541, Department of Genetic Engineering, Sungkyunkwan University, Suwon 16419, 3 Institute for Bio-Medical Convergence, International St. Mary’s Hospital and College of Medicine, Catholic Kwandong University, Incheon 22711, 4 College of Pharmacy, Duksung Women’s University, Seoul 01369, Republic of Korea 1 2
Abstract Cordyceps bassiana is one of Cordyceps species with anti-oxidative, anti-cancer, anti-inflammatory, anti-diabetic, anti-obesity, anti-angiogenic, and anti-nociceptive activities. This mushroom has recently demonstrated to have an ability to reduce 2,4-dinitrofluorobenzene-induced atopic dermatitis symptoms in NC/Nga mice. In this study, we further examined phytochemical properties of this mushroom by column chromatography and HPLC analysis. By chromatographic separation and spectroscopic analysis, 8 compounds, such as 1,9-dimethylguanine (1), adenosine (2), uridine (3), nicotinamide (4), 3-methyluracil (5), 1,7-dimethylxanthine (6), nudifloric acid (7), and mannitol (8) were identified from 6 different fractions and 4 more subfractions. Through evaluation of their anti-inflammatory activities using reporter gene assay and mRNA analysis, compound 1 was found to block luciferase activity induced by NF-kB and AP-1, suppress the mRNA levels of cyclooxygenase (COX)-2 and tumor necrosis factor (TNF)-a. Therefore, our data strongly suggests that compound 1 acts as one of major principles in Cordyceps bassiana with anti-inflammatory and anti-atopic dermatitis activities. Key Words: Cordyceps bassiana, Phytochemical study, 1,9-dimethylguanine, NF-kB, AP-1, Anti-inflammatory activity
INTRODUCTION
C. bassiana was described in China on a carpenterworm larva and connected to unequivocally B. bassiana (Rehner et al., 2011). More recently, C. bassiana was found in Korea and the evidence of the teleomorph-anamorph connection in the Cordyceps specimen was provided, in addition to in vitro production of C. bassiana (Hyun et al., 2013). By systematic analysis of the pharmacological activities of the Cordyceps species to date, anti-oxidative, anti-cancer, anti-inflammatory, anti-diabetic, anti-obesity, anti-angiogenic, and anti-nociceptive activities have been demonstrated (Ng and Wang, 2005; Zhou et al., 2009). Recently, extracts of the artificially cultivated C. bassiana have been reported to inhibit the production of nitric oxide, interleukin (IL)-12, and interferon (IFN)-γ in lipopolysaccharide (LPS)-stimulated macrophages, splenic lymphocytes (Byeon et al., 2011a, 2011b), as well as ameliorating atopic dermatitis symptoms in 2,4-dinitrofluorobenzene-treated NC/Nga mice (Wu et al., 2011). In spite
Cordyceps species are insect-born mushrooms that have been known to possess a variety of biological material. As such, the Cordyceps species has been used for a long time in Chinese and Korean traditional folk medicine as a tonic for longevity, endurance and vitality, and it has been traditionally used as a therapeutic remedy for various diseases, including eczema, skin diseases, chronic bronchitis, asthma, and tuberculosis (Ng and Wang, 2005; Zhou et al., 2009). Cordyceps bassiana is known to be a teleomorph (sexually reproducing form) of Beauveria bassiana. B. bassiana is a ubiquitous fungal pathogen of insect and fungal biocontrol agents widely used against insects (Rehner et al., 2011) and widely used as a medicine for the treatment of infantile convulsions, epilepsy, stroke, sore throat, and many sorts of wounds before being used for insect biocontrol (Madsen et al., 2007). In contrast,
Received Mar 21, 2016 Revised May 5, 2016 Accepted Jun 20, 2016 Published Online Aug 19, 2016
Open Access https://doi.org/10.4062/biomolther.2016.063 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
*Corresponding Authors
E-mail: [email protected] (Shim SH), [email protected] (Cho JY) Tel: +82-2-901-8774 (Shim SH), +82-31-290-7868 (Cho JY) Fax: +82-2-901-8386 (Shim SH), +82-31-290-7870 (Cho JY) † The first three authors contributed equally to this work.
www.biomolther.org
Copyright © 2017 The Korean Society of Applied Pharmacology
165
Biomol Ther 25(2), 165-170 (2017)
A
of its valuable pharmacological activities, no reports on the chemical constituents of C. bassiana have been published to date. Simultaneously, we are trying to develop an anti-atopic dermatitis remedy with this species, and have also performed research to dissect the chemical constituents of C. bassiana in order to identify the active components. Herein, isolation, structure determination, and anti-inflammatory activities of the 8 major compounds from a water extract of C. bassiana fruit body displaying anti-atopic dermatitis activity are described.
MATERIALS AND METHODS Materials
Artificially cultivated Cordyceps bassiana (C. bassiana) (Fig. 1A) were purchased from Mush-Tech (Gangwon, Korea), identified by Prof. Jae Mo Sung (Kangwon National University, Chuncheon, Korea). A voucher specimen (No. 1001) was deposited in the herbarium of Daewoong Pharmaceutical Co. Ltd. (Yongin, Korea). Lipopolysaccharide (LPS, E. coli 0111:B4) and (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bay 11-7082 (BAY), SB203580 (SB), and U0126 (U0) were obtained from Calbiochem (La Jolla, CA, USA). Luciferase constructs containing binding promoters for NF-kB and AP-1 were gifts from Prof. Chung Hae Young (Pusan National University, Pusan, Korea) and Man Hee Rhee (Kyungpook National University, Daegu, Korea). Foetal bovine serum and RPMI 1640 were obtained from GIBCO BRL (Grand Island, NY, USA). RAW264.7 and HEK293 cells were purchased from ATCC (Rockville, MD, USA). All other chemicals were of Sigma grade. A powdered extract of C. bassiana was provided by Daewoong Pharmaceutical Co. Ltd (Yongin, Korea).
Artificially cultivated fruit body of C. bassiana
B Luciferase activity (fold increase)
50
30 20 10
**
**
*
**
0 Nor
0 50 100 NF- B-Luc
Nor
0 50 100 AP-1-Luc
Fig. 1. A photo of an artificially cultivated fruit body of Cordyceps bassiana and the inhibitory activity of its water extract on AP-1 and NF-kB-mediated luciferase. (A) A photo of an artificially cultivated fruit body of Cordyceps bassiana. (B) HEK293 cells co-transfected with plasmid constructs NF-kB-Luc or AP-1-Luc (each 1 mg/ml) and b-gal (as a transfection control) were treated with the water extract of Cordyceps bassiana in the presence or absence of PMA (100 nM). Luciferase activity was measured using a luminometer. *p
